CN106518831B - A kind of plant proanthocyanidin dimer, tripolymer quick separating preparation method - Google Patents
A kind of plant proanthocyanidin dimer, tripolymer quick separating preparation method Download PDFInfo
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- CN106518831B CN106518831B CN201610946727.8A CN201610946727A CN106518831B CN 106518831 B CN106518831 B CN 106518831B CN 201610946727 A CN201610946727 A CN 201610946727A CN 106518831 B CN106518831 B CN 106518831B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
- C07D311/60—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2
- C07D311/62—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2 with oxygen atoms directly attached in position 3, e.g. anthocyanidins
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Abstract
The invention discloses a kind of plant proanthocyanidin dimer, tripolymer quick separating preparation methods, the ethanol water solution extract that will be enriched in proanthocyanidin plant material crosses gel column after liquid-liquid extraction, it is detached again through FPLC or HSCCC, respectively obtains proanthocyanidin dimer, tripolymer ingredient.The proanthocyanidin dimer and tripolymer that the technique of the present invention is suitable under separate sources isolate and purify, and have universality.Proanthocyanidin dimer that the process of the present invention obtains, tripolymer purity are high, method security is strong, it is easy to operate, take short, production cost is low, it is environmental-friendly, meet Green Chemistry standard, be suitable for industrialized production, there is great commercial value.
Description
Technical field
The invention belongs to flavan-3-alcohol dimer, tripolymer quick separating preparation method technical fields, and in particular to a kind of
Plant proanthocyanidin dimer, tripolymer quick separating preparation method.
Background technology
Proanthocyanidin is the general name for the major class polyphenol compound that nature is widely present, be a kind of Flavanol monomers and
Its polymeric polyphenolic substance.Polyphenols has stronger oxidation resistance and other biological activity, is beneficial to human body
Health, Procyanidins are the substances that content is most abundant in plant polyphenol compound, are increasingly subject to the concern of people.
Proanthocyanidin has compared with VE, Vc, the higher bioavailability of carrotene, and can and VEGenerate synergistic oxidation effect.
Proanthocyanidin was mainly used for medicine, health products, cosmetics and functional polymer functional material etc. in recent years.However, needle
Research effect is general in terms of being isolated and purified to proanthocyanidin, mainly carries out classification separation to it, prepares oligomeric proanthocyanidins.
Rie Kusano in 2011 et al. obtain proanthocyanidin sterling from acacia mearnsii extract, but complex process and yield it is low.Grape
Isolating and purifying for seed proanthocyanidin is easy compared with acacia mearnsii proanthocyanidin, and HSCCC isolates and purifies Proanthocyanidins from Grape Seeds technology at present
It is increasingly mature.Sun Baoshan in 2015 et al. prepares 77% catechin, 93% epicatechin, 85% table by HSCCC from grape pip
Catechin and gallate etc..Black wattle bark proanthocyanidin ingredient is more complicated, finds that simple, suitable industrialized separation is pure
Change method.Therefore, it is most important to find a kind of method that can be detached very well to different material.
Invention content
Goal of the invention：For the deficiencies in the prior art, the object of the present invention is to provide a kind of plant proanthocyanidins
Dimer, tripolymer quick separating preparation method, have it is simple for process and at low cost, can industrialize, have the characteristics that universality.
Technical solution：In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is：
It is former to will be enriched in proanthocyanidins plant for a kind of plant proanthocyanidin dimer, tripolymer quick separating preparation method
Feed powder is broken, after sieving, with ethanol water ultrasonic wave assisted extraction, after extracting solution filters, recycling ethyl alcohol is concentrated under reduced pressure, freezing is dry
It is dry to constant weight, obtain extract；Said extracted object is dissolved in water, with petroleum ether extraction, aqueous layer with ethyl acetate extraction obtains acetic acid second
Ester layer substance；Ethyl acetate layer substance is dissolved in ethyl alcohol, sample is crossed into gel column, isocratic elution is carried out with ethanol water,
Eluent is collected, recycling ethyl alcohol is concentrated under reduced pressure, freeze-drying is to constant weight to get proanthocyanidin first product；Proanthocyanidin first product is passed through
Fast protein liquid chromatography（FPLC）Or high speed adverse current chromatogram（HSCCC）Separation to get to proanthocyanidin dimer, trimer at
Point.
The plant proanthocyanidin dimer, tripolymer quick separating preparation method, are as follows：
1）Plant material is crushed, crosses 30-60 mesh sieve, with ethanol water ultrasonic wave assisted extraction, obtain extracting solution into
Row rotary evaporation concentrates；
2）Above-mentioned concentrating sample is extracted into obtain ethyl acetate layer through petroleum ether, ethyl acetate successively, is concentrated under reduced pressure, freezing is done
It is dry to obtain acetic acid ethyl ester extract；
3）Above-mentioned acetic acid ethyl ester extract powder is dissolved in ethyl alcohol, after the gel column pre-processed, is carried out with ethyl alcohol
Isocratic elution collects different component；
4）The component being collected into is subjected to liquid-phase chromatographic analysis, same composition is merged, merge component by FPLC or
HSCCC is further purified to get proanthocyanidin dimer, tripolymer sterling.
The plant proanthocyanidin dimer, tripolymer quick separating preparation method, plant material includes acacia mearnsii, Portugal
Grape seed.
Step 1）In, it is extracted as solvent using 30% ~ 80% ethyl alcohol, liquid ratio 1:5~1:15, ultrasonic time is
10 ~ 60min, supersonic frequency 60kHz, 35 ~ 70 DEG C of Extracting temperature.
Step 3）In, acetic acid ethyl ester extract is filtered through gel column Sephadex G-25 or Sephadex LH-20, is used
70% ~ 100% isoconcentration ethyl alcohol carries out isocratic elution, and elution flow rate is 0.5 ~ 1.5mL/min, and 10-50mL is as a component
It is collected.
Step 4）In, FPLC preparative separations select first alcohol and water for mobile phase, progress gradient elution, first under 6 column volumes
Determining alcohol is linearly increasing with 5% to 60%.
Step 4）In, HSCCC dicyandiamide solutions are acetate-methanol-water or n-butanol-acetate-methanol-water.
The proanthocyanidin dimer of the present invention, tripolymer quick separating preparation method, using ultrasonic wave auxiliary extraction technology
Proanthocyanidin is extracted, gained extracting solution is concentrated under reduced pressure, is freeze-dried.Extract petroleum ether is extracted with water, water layer acetic acid second
Ester is extracted.Ethyl acetate layer is concentrated under reduced pressure, is freeze-dried.Above-mentioned ethyl acetate layer substance is dissolved in ethyl alcohol, by sample
Gel column Sephadex G-25 and Sephadex LH-20 are crossed, are that mobile phase carries out isocratic wash with 70% ~ 100% ethanol water
De-, elution flow rate is 0.5 ~ 1.5mL/min, collects eluent, recycling ethyl alcohol is concentrated under reduced pressure, freeze-drying is to constant weight to get original
Anthocyanidin first product；Above-mentioned proanthocyanidin first product is spent through fast protein liquid chromatography and high speed adverse current chromatogram separation to get to original
Pigment dimer, tripolymer.
Advantageous effect：Compared with prior art, proanthocyanidin dimer of the invention, tripolymer quick separating preparation side
Method, using ultrasonic wave assisted extraction, using ethanol water as solvent, sample crosses gel column Sephadex after simple extraction and separation
G-25 and Sephadex LH-20, mobile phase select ethanol water.Using technology nonhazardous substance in operation
Addition, high safety are easy to operate, it is only necessary to cross a gel column, take short, separative efficiency height, proanthocyanidin dimer, three
The purity and yield of aggressiveness are higher.2）Use ethanol water for Extraction solvent in preparation process, it is water-soluble with ethyl alcohol in column separation process
Liquid is mobile phase, and solvent is cheap and easily-available and environmental-friendly, therefore is more suitable for industrialized production proanthocyanidin, the isolation and purification method
With very high commercial value.
Description of the drawings
Fig. 1 is Black Wattle extract ethyl acetate extract layer liquid chromatogram；
Fig. 2 is black wattle bark proanthocyanidin first product LC-MS analysis chart；Wherein, For dimer,For tripolymer；
Fig. 3 is acacia mearnsii proanthocyanidin first product through dimer LC-MS analysis chart isolated FPLC；
Fig. 4 is black wattle bark proanthocyanidin high speed adverse current chromatogram preparative separation figure；
Fig. 5 is acacia mearnsii proanthocyanidin first product through tripolymer LC-MS analysis chart isolated FPLC；
Fig. 6 is the acetic acid ethyl ester extract liquid chromatogram of grape seed extract；
Fig. 7 is the LC-MS analysis chart that grape pip acetic acid ethyl ester extract detaches sample through gel filtration column.
Specific implementation mode
With reference to specific drawings and examples, the invention will be further described.
Embodiment 1
By black wattle bark raw materials of the 10g through pulverizing and sieving, according to solid-liquid ratio 1:10 are added 60% ethyl alcohol of 100mL, 50
Ultrasonic wave assisted extraction 30min is used at DEG C, and extracting solution is obtained after filtering.Extract is dissolved in 200mL by extracting solution concentrated by rotary evaporation
In water.100mL petroleum ether extractions are added in aqueous solution（Three times）, obtain water layer.Water layer is extracted with 600mL ethyl acetate（Three times）, obtain
Ethyl acetate layer.Ethyl acetate layer is spin-dried for and is freeze-dried to obtain Black Wattle ethyl acetate extract, the liquid phase color of ingredient
Spectrum analysis the result is shown in Figure 1.As shown in Figure 1, black wattle bark extract complicated component after ethyl acetate extracts, liquid phase figure occur
Folded peak then illustrates ethyl acetate layer, and there are the isomers of proanthocyanidin oligomer, increase unification in acacia mearnsii proanthocyanidin
That closes object isolates and purifies difficulty.
2g Black Wattle acetic acid ethyl ester extracts are dissolved in about 20mL ethyl alcohol, 0.45 μm of filter membrane, spare is crossed.The sample of dissolving
Product cross Sephadex LH-20 gel columns, and isocratic elution is carried out with a concentration of 100% ethyl alcohol.Component is collected with 10mL/ pipes, it will
Obtained component carries out liquid phase analysis, merges same composition.Component will be merged and carry out liquid matter analysis, primarily determine its structure, as a result
See Fig. 2.As shown in Figure 2, proanthocyanidin first product is crossed into sephadex column, can be by proanthocyanidin according to not with ethyl alcohol isocratic elution
It is detached with the degree of polymerization, the acacia mearnsii proanthocyanidin dimer of 60% purity can be made, 47% purity acacia mearnsii original pattern
Plain dimer, 50% purity acacia mearnsii proanthocyanidin tripolymer, which is further purified convenient for follow-up monomer.
The acacia mearnsii proanthocyanidin dimer for being 60% by 0.1g purityIt is dissolved in about 10mL water, sample dissolution passes through
FPLC is isolated and purified, and mobile phase is methanol and water, and stationary phase is XBP C18 semi-preparative columns, and elution process is 5% ~ 60% methanol ladder
Degree elution.Applied sample amount is 2mL, 1 ~ 1.5mL/min of flow velocity.Merge same composition.Component will be merged and carry out liquid matter analysis, as a result seen
Fig. 3.From the figure 3, it may be seen that FPLC can efficiently separate purifying sephadex column separation product dimer, proanthocyanidin dimer is made
Purity is up to 92%.
The acacia mearnsii proanthocyanidin dimer for being 60% by 0.1g purityIt is dissolved under about 20mL adverse current chromatograms in phase, dissolves
Sample is isolated and purified by HSCCC, and dicyandiamide solution is acetate-methanol-water.Upper phase is pumped into 15 ~ 25mL/min, rotating speed
750 ~ 900r/min is pumped into mobile phase with 1 ~ 3mL/min.It is collected by peak and merges same composition.Component will be merged and carry out liquid matter point
Analysis, proanthocyanidin dimer purity are shown in Fig. 4 up to 92%, HSCCC preparative separation results.As shown in Figure 4, HSCCC can be efficiently separated
Sephadex column separation product is purified, 3 components progress liquid phase analysis are collected according to the online collection of illustrative plates of Fig. 4 and merges target group
Point, it is convenient for the collection of target components.
The acacia mearnsii proanthocyanidin tripolymer sample that 0.1g purity is about 50% is dissolved in 10mL water, sample dissolution passes through
FPLC is isolated and purified, and mobile phase is methanol and water, and stationary phase is XBP C18 semi-preparative columns, and elution process is 5% ~ 60% methanol ladder
Degree elution.Applied sample amount is 2mL, 1 ~ 1.5mL/min of flow velocity.Merge same composition.Component will be merged and carry out liquid matter analysis, as a result seen
Fig. 5.As shown in Figure 5, FPLC can efficiently separate purifying sephadex column separation product tripolymer, and proanthocyanidin tripolymer is made
Purity is up to 93%.
Embodiment 2
By grape seed raw materials of the 10g through pulverizing and sieving, according to solid-liquid ratio 1:10 are added 100mL60% ethyl alcohol, are adopted at 50 DEG C
With ultrasonic wave assisted extraction 30min, extracting solution is obtained after filtering.Extract is dissolved in 200mL water by extracting solution concentrated by rotary evaporation,
100mL petroleum ether extractions are added in aqueous solution（Three times）, obtain water layer.Water layer is extracted with 600mL ethyl acetate（Three times）, obtain acetic acid second
Ester layer.Ethyl acetate layer is spin-dried for and obtains grape pip ethyl acetate extract, the liquid-phase chromatographic analysis of ingredient after being freeze-dried
As a result see Fig. 6.It will be appreciated from fig. 6 that grape pip crude extract obtains grape pip oligomer after ethyl acetate extracts, complicated component needs
It further to isolate and purify.
2g grape pip acetic acid ethyl ester extracts are dissolved in 20mL ethyl alcohol, 0.45 μm of filter membrane, spare is crossed.The sample mistake of dissolving
Sephadex G-25, with a concentration of 100% ethyl alcohol isocratic elution.Component is collected with 10mL/ pipes, obtained component is subjected to liquid phase
Analysis merges same composition.Component will be merged and carry out liquid matter analysis, its structure is primarily determined, as a result see Fig. 7.As shown in Figure 7,
Proanthocyanidins from Grape Seeds first product can effectively be divided grape pip oligomer through sephadex column filtering, with ethyl alcohol isocratic elution
From the Proanthocyanidins from Grape Seeds dimer of 80% or more purity being made, method is simple and reproducible, illustrates sephadex
Column filtering cooperation ethyl alcohol isocratic elution is a kind of ideal proanthocyanidin dimer, tripolymer isolation and purification method.
Claims (5)
1. a kind of plant proanthocyanidin dimer, tripolymer quick separating preparation method, it is characterised in that：It will be enriched in proanthocyanidin
The proanthocyanidin acetic acid ethyl ester extract of class plant material is dissolved in ethyl alcohol, and sample is crossed gel column, is carried out with ethanol water
Isocratic elution collects eluent, recycling ethyl alcohol is concentrated under reduced pressure, freeze-drying is to constant weight to get proanthocyanidin first product；By former pattern
Plain first product is through fast protein liquid chromatography or high speed adverse current chromatogram separation to get to proanthocyanidin dimer, tripolymer ingredient；Its
In, plant material is acacia mearnsii, and the quick separating preparation of dimer, tripolymer is as follows：
1) plant material crushed, cross 30-60 mesh sieve, with ethanol water ultrasonic wave assisted extraction, obtained extracting solution and revolved
Turn to be concentrated by evaporation；
2) above-mentioned concentrating sample is extracted into obtain ethyl acetate layer through petroleum ether, ethyl acetate successively, is concentrated under reduced pressure, is freeze-dried
Acetic acid ethyl ester extract；
3) above-mentioned acetic acid ethyl ester extract powder is dissolved in ethyl alcohol, after the gel column pre-processed, is carried out with ethyl alcohol isocratic
Different component is collected in elution；
4) by the component being collected into carry out liquid-phase chromatographic analysis, same composition is merged, merge component by FPLC or HSCCC into
One step purifies to get proanthocyanidin dimer, tripolymer sterling.
2. plant proanthocyanidin dimer according to claim 1, tripolymer quick separating preparation method, feature exist
In：In step 1), extracted as solvent using 30%~80% ethyl alcohol, liquid ratio 1:5~1:15, ultrasonic time 10
~60min, supersonic frequency 60kHz, 35~70 DEG C of Extracting temperature.
3. plant proanthocyanidin dimer according to claim 1, tripolymer quick separating preparation method, feature exist
In：In step 3), acetic acid ethyl ester extract is filtered through gel column Sephadex G-25 or Sephadex LH-20, using 70%
~100% isoconcentration ethyl alcohol carries out isocratic elution, and elution flow rate is 0.5~1.5mL/min, and 10-50mL is as a component
It is collected.
4. plant proanthocyanidin dimer according to claim 1, tripolymer quick separating preparation method, feature exist
In：In step 4), FPLC preparative separations select first alcohol and water for mobile phase, progress gradient elution, methanol concentration under 6 column volumes
It is linearly increasing with 5% to 60%.
5. plant proanthocyanidin dimer according to claim 1, tripolymer quick separating preparation method, feature exist
In：In step 4), HSCCC dicyandiamide solutions are acetate-methanol-water or n-butanol-acetate-methanol-water.
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